| Project/Area Number |
18310053
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Environmental technology/Environmental materials
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| Research Institution | Tokyo University of Agriculture and Technology |
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Principal Investigator |
RYOICHI Sato Tokyo University of Agriculture and Technology, Institute of Symbiotic Science and Technology, Associate Professor (30235428)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥17,630,000 (Direct Cost: ¥15,800,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2007: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2006: ¥9,700,000 (Direct Cost: ¥9,700,000)
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| Keywords | biotechnology / protein / microorganism / Bacillus thruingiensis / insecticide |
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| Research Abstract |
Bacillus thuringiensis produces Cry toxins, insecticidal proteins which are induced production at the time of the sporulation. It is theoretically possible to make activity improved toxin by increasing affinity of the toxin to the receptor altering amino-acid sequences especially of the receptor-binding region of the toxin. In this research, we used a phage display system, which was developed in our laboratory, and tried to achieve the directed evolution of Cry toxins to increase the affinity to the receptor. To clarify the receptor binding region, first we constructed 13 cysteine-replaced mutant toxin DNA by site-directed mutagenesis. After production of mutant toxins, we utilized the thiol of the cysteine to be bound with some molecules as inhibitors of receptor-binding, and analyzed these mutants whether inhibition occurred or not. From these experiments we concluded that face made by loop 2, loop 3 and loop alpha 8 is important as a receptor binding site. Next we constructed 11 phage libraries displaying mutant Cry toxin with changed amino acids at loop 2, loop 3 or loop alpha 8. Selection of mutant Cry toxin by bio-panning or magnetic beads system was conducted. From some libraries, concentration of some mutant toxin displaying phages was observed. Although we are now confirming the actual insecticidal activity and improvement of binding affinity to the receptor of each mutant toxin, we are expecting that those mutants have improved insecticidal activities.
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